Genetic and anthropological investigations on ancient skeletal remains recovered in Milan, from the Roman period to the Contemporary Age

The Milan project arises to describe the life and evolution of the populations living in the urban area of Milan across two millennia (from the Roman Imperial Age to Contemporary times) through a multidisciplinary approach involving archaeology, anthropology and genetics. This research project represented an articulated pilot study carried out to investigate a selection of this ancient material combining anthropology and genetics. In particular, this was the first attempt performed to assess the feasibility of reconstructing the history of Milan through DNA analysis. To this purpose, ten intriguing skeletal remains excavated from seven different sites and belonging to different historical periods were selected and submitted to genetic and osteological analyses. In particular, DNA extraction, genetic sex typing and mitochondrial DNA analysis were performed applying protocols and procedures commonly used in cases of highly degraded samples. Mitochondrial DNA studies were carried out applying two different protocols in order to recover the complete mitogenome (mitoTiling protocol) and enrich samples in sequences of interest (PEC protocol). Finally, to evaluate how the different archaeological contexts could influence bone tissue appearance and to verify the presence of a correlation with DNA content, macroscopic and microscopic analyses were performed. The results highlighted the great potential of the approach applied, providing intriguing data concerning the biological profiles of each individual. In particular, anthropology provided interesting results, specifically about stress markers and pathology, highlighting the difficult living conditions and the situation of extreme poverty of people during these historical periods. In addition, genetics was able to enrich biological profiles supplying interesting information about the geographic origin, also for some of the cases for which no anthropological estimation could be performed. For this reason, the methods used to evaluate genetic sex and mitochondrial DNA were proved to be efficient in the recovery of these information from such ancient material. In particular, PEC protocol was considered very efficient in the enrichment of highly degraded samples. Moreover, genetic analyses pointed out degraded DNA in the bone samples characterised by very short fragments length, low amounts of DNA and peculiar damage patterns. Both time since deposition and environmental conditions influenced DNA preservation in the bone samples. Similar results were obtained only from the microscopic analysis of calcified and decalcified bone thin sections. In spite of this similarity, no relation between bone structure appearance and genetic data was detected, even if the small sample size limits the final conclusion about a possible correlation. Moreover, differences identified between macroscopic and microscopic preservation highlighted the importance to include the histological analysis, in particular of decalcified thin sections, in the evaluation of bone tissue appearance. Finally, these preliminary results supported the possibility to carry on further analyses on this skeletal population. For this reason, the most intriguing samples will be further analysed in the Max Planck Institute for the Science of Human History in Jena (Germany), applying specific protocols and techniques used in the ancient DNA field, in order to investigate the DNA content, both human and microbial, and to assess the feasibility to analyse other interesting genetic markers. In addition, as a future perspective, an increase in the number of individuals collected from each site could be planned to provide a portrait of the evolution of the Milan population over time.